In deuterostomes, endoderm and mesoderm initially are cospecified as endomesoderm, and later subdivided into definitive endoderm and mesoderm by cell-cell signaling. Ectoderm depends upon signals from the endomesoderm for its axial properties. In the first phase of this project we established beta-catenin as the earliest known molecule required for specification of endomesoderm in the sea urchin embryo, a prototype deuterostome. We established that a Delta-Notch signal is responsible for the subdivision of endomesoderm into endoderm and mesoderm, and we collaborated in the assembly of a large provisional Gene Regulatory Network (GRN) model for endomesoderm cell specification. This proposal has three overriding specific aims that extend our knowledge of the gene regulatory network concept. I. The micromere gene regulatory network: The aim is to better understand the activation of the micromere GRN including the release of three distinct inductive signals from micromeres during the first 7-9 cleavages. Our first specific aim is to establish (i) how components in the egg vegetal cortex activate the network; (ii) the identity and function of the early endoderm induction signal (iii) how wnt 8, an autocrine signal in micromeres, is activated and regulates downstream network function, and (iv) how micromere GRN transcription factors set up the signal to begin an epithelial-mesenchymal transition. II. The endomesoderm gene regulatory network: Preliminary data suggests there are three distinct Notch signals necessary for endomesoderm specification during the first 10 cleavages. The first signal is from micromere to macromere to separate mesoderm from endomesoderm. The second Notch signal appears to reinforce the nascent mesoderm-endoderm boundary and the third Notch signal appears to occur later in the endoderm. This aim asks how the sequential use of the Notch signaling pathway fits into the gene regulatory network spatially and temporally in three locations at three different times, all within the first 10 cleavages. Ill. The oral-aboral gene regulatory network: This aim focuses on how oral-aboral specification is induced by endomesoderm derivatives, through BMP and p38 MAP kinase, and how the oral aboral gene regulatory network is organized. Ultimately, the oral-aboral ectoderm provides positional cues to the skeletogenic mesenchyme in the blastocoel beneath this epithelium. This network eventually will enable us to explore how positional cues are produced and displayed for later morphogenetic patterning.